Location: Floral and Nursery Plants Research
2020 Annual Report
Objectives
Objective 1: Determine whether parasitic genes previously isolated from the root lesion nematodes associated with hairy roots of soybean can confer nematode resistance in lilies, and use the biolistic approach to transform lilies and develop cultivars that are resistant to this migratory nematode pest. [NP301, C1, PS1A, PS1B; C3, PS3B]
Sub-objective 1.1: Optimize biolistic-mediated transformation of lilies to increase the frequency of transformation events.
Sub-objective 1.2: Compare levels of uidA reporter gene expression in roots of lilies transformed with either the CaMV 35S, maize Ubi1, Arabidopsis UBQ3, or Gladiolus GUBQ1 promoters to determine the promoter that directs the highest levels of transgene expression in roots.
Sub-objective 1.3: Transform Easter lilies with ds-FAR1 and ds-GPX genes and evaluate them for resistance to P. penetrans.
Objective 2: Identify parasitic genes that can be used to confer burrowing nematode resistance in anthuriums using genomic analysis techniques, develop a system of delivery to incorporate the identified genes into resistant cultivars of anthuriums, and evaluate the genes. [NP301, C1, PS1A, PS1B; C3, PS3B]
Sub-objective 2.1: Develop carrot hairy roots containing unc87 and FAR1 from R. similis as dsRNA constructs and screen the carrot hairy roots for resistance to R. similis.
Sub-objective 2.2: Transform Anthurium with ds-unc87 and ds-FAR1 gene constructs and screen Anthurium lines for resistance.
Approach
Transgenic Easter lilies and anthuriums that contain dsRNA constructs of parasitic genes will be developed and plants screened for resistance to the migratory nematodes that infect them.
The biolistic transformation system for Easter lilies will be optimized. Four promoters, CaMV 35S, maize Ubi1, Arabidopsis UBQ3, and Gladiolus GUBQ1, will be compared in Easter liies to determine which promoters express highly in roots where the migratory nematode Pratylenchus penetrans infects. Lilies will be transformed with either the parasitic gene ds-FAR1 or ds-GP, and transgenic plants will be screened for resistance to P. penetrans infection.
Two genes, unc87 and FAR1, will be isolated from a transcriptome of Radopholus similis, the burrowing nematode, using BLAST to search for homology to these same genes in other migratory nematodes. Hairy roots of carrots and anthuriums containing dsRNA constructs of the R. similis unc87 and FAR1 will be developed and screened for resistance.
Progress Report
This is the final report for the project 8020-21000-068-00D which terminated in December 2019, following the retirement of the SY in April 2019. There is no corresponding new Project. All planned experiments were completed prior to termination.
Substantial progress was made over the life of this project. Easter lilies were transformed with the dsFar and dsGP genes from Pratylenchus penetrans; however, they did not show resistance to the nematode when challenged in vitro with P. penetrans. Four constitutive promoters - CaMV 35S, Gladiolus ubiquitin, Arabidopsis ubiquitin, and maize ubiquitin - were tested in transgenic lilies containing the GUS reporter gene under each promoter. Both the CaMV 35S and Gladiolus ubiquitin promoter showed higher levels of GUS expression than the other two promoters. Leaves with the CaMV 35S promoter showed more GUS expression than leaves with the Gladiolus ubiquitin promoter. In addition, we tested the effects of several factors on lily transformation efficiency and found that bombarding cells once with the gene gun is comparable to bombarding twice. We also found that increasing mannitol concentration (which protects cells from bursting) did not enhance the transformation frequency. We found that bulb scales that were preconditioned on BA and NAA with a low level of picloram had a lower rate of transformation, but higher regeneration, than bulb scales that were preconditioned with higher levels of picloram alone. Finally, we found that using smaller gold particles did not improve transformation frequencies.
In a collaborative project with ARS scientists in Hawaii, two transcriptomes were made using RNA from burrowing nematodes. These nematodes have some of the same parasitism genes as P. penetrans that affect plant roots, but also have unique genes that may serve as targets for specifically controlling burrowing nematodes. We created carrot hairy roots containing ds-PAT, ds-unc, ds-pas, and ds-VAP from the burrowing nematode transcriptome. These transformants were verified by PCR and sent to the ARS collaborators in Hawaii for use in their studies on anthurium.
A patent was granted in September 2019 (US 10,407,681 B2) for plant-mediated silencing (RNAi) of proteins utilizing dsRNA to control parasitic nematodes, including P. penetrans. The patent describes specific synthetic dsRNAs that induce RNAi in the target nematodes, along with the methods of delivering the dsRNAs to the nematode, including feeding on transgenic plants that contain the dsRNA.
Accomplishments
